A multifactorial role for P. falciparum malaria in endemic Burkitt's lymphoma pathogenesis

Abstract

Endemic Burkitt's lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of P. falciparum malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that P. falciparum targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria.

Figure 1. P. falciparum extracts stimulate expression of AID mRNA in normal tonsil B cells.

Tonsils lymphocytes were isolated and incubated with the components indicated in the Figure for 3 (blue), 5 (red) or 7 (green) days. After the noted incubation times, B cells were isolated and analyzed for AID mRNA expression. A. Induction of AID mRNA requires a combination of signals from IL-4, CD40 ligand and CpG. B. P. falciparum extracts stimulate AID mRNA expression more effectively than CpG. AU – arbitrary units. The level of AID mRNA is expressed relative to that of β-actin.

Figure 2. P. falciparum extracts stimulate AID expression at levels equivalent to CpG combined with surface Ig cross-linking.

The assay was performed after incubation for 5 days as described in Figure 1.

Figure 3. P. falciparum extracts stimulate expression of AID protein in normal tonsil B cells.

The same protocol was followed as described in Figure 1, except the cells were analyzed by staining for AID protein expression and FACS analysis after a 5 day incubation period. - Flow cytometry histograms of cell populations demonstrate equivalent levels of AID expression in B cells activated by CpG or P. falciparum extracts. MFI  =  Mean fluorescence intensity. A. and C. Percentage of all B cells expressing AID (B) and percent of B cells expressing high levels of AID (C) suggest equal levels of AID expression in B cells activated by CpG or P. falciparum extract. We observed two overlapping populations of cells staining positive for AID. An arbitrary gate imposed on the brighter population defined high level expressers. The same gate was applied to all samples.

Figure 4. Hemozoin is taken up by B cells and activates AID expression.

A. Hemozoin (sHz) (red) and CpG (green) were incubated with two different B cell lines. Note presence of red granules inside both cell types. BL2 is an EBV negative BL line and IM171 a spontaneous EBV positive lymphoblastoid line. B. Hemozoin stimulates expression of AID mRNA to an equivalent level to that obtained with surface Ig cross-linking and CpG. The stimulation was independent of hemozoin being complexed with parasite DNA. The same protocol was followed as described in Figure 1. The cells were analyzed after a 5 day incubation period.

Figure 5. Higher levels of AID and c-myc mRNA in tonsil GC B cells from individuals infected with P. falciparum malaria compared to controls.

A. AID mRNA expression is significantly increased in GC B cells from the malaria tonsils. (** p = 0.005). B. c-myc RNA is significantly elevated in GC B cells from some malaria tonsils. (* p = 0.031). AID and c-myc mRNA levels were normalized to β-actin and the level in the GC population is expressed relative to that in a standard naïve B cell population (calibrator) isolated from a single Boston tonsil.

Figure 6. Higher levels of EBV infected cells in tonsil GCs from individuals infected with P. falciparum malaria compared to controls.

A. The percentage of B cells (CD19+) is unchanged. (ns p = 0.18). B. The percentage of GC B cells (CD10+) is significantly elevated in the malaria tonsils (*** p<0.001). C. The frequency of GC B cells latently infected with EBV is dramatically increased in the malaria tonsils (*** p = 0.001). For details see Table 1. D. The level of EBV infected GC B cells and AID expression do not directly correlate.

Figure 7. c-myc is expressed in GC B cells.

A. Flow cytometric analysis demonstrating c-myc expression in tonsil GC cells (CD10+). The arrow indicate the CD10+, c-myc positive GC population. B. Western blot analysis confirming c-myc expression in 3 independent tonsil GC B cell preparation (GC1-3). Raji and Rael are EBV positive BL cell lines. The molecular weight in KD is shown to the left. C. ImageStream analysis of c-myc positive tonsil GC B cells. Staining for a known nuclear protein bcl-6 is shown for comparison. N.B. For this study only Boston control tonsils were used.